Implantable and biocompatible gellable composition

ABSTRACT

The invention concerns an aqueous biocompatible gel composition of polysaccharides characterized in that it comprises a mixture of at least two polysaccharides one of which is capable of being gelled by chemical process and the other by heat process and the use of such a composition for preparing an implant. The inventive composition is characterized in that it further contains biological tissue extracts and/or an active pharmaceutical principle alone or combined.

The present invention relates to the formulation and the shaping ofbiocompatible gellable compositions for producing implants.

In the field of the healing of tissues such as cartilage or bonetissues, it is often necessary to resort to chondrocyte transplants.

These chondrocyte transplants use, to date, cell suspensions which aresometimes difficult to use and, as a result, there is a need for aproduct or for means for carrying out these chondrocyte transplantsusing solid materials exhibiting mechanical characteristics which alloweasy handling.

Biodegradable materials are known, namely, for example, natural polymerssuch as celluloses, methylcelluloses, gelatins, caseins, chitosans,polylysines or polysaccharides.

The properties, for example gelling properties, of some of thesebiomaterials, such as polysaccharides, which make it possible to producesolid products, and their biocompatibility which is sufficient tomaintain cell viability, are also known. It is thus known practice tokeep cells alive in media containing natural polysaccharides, and theinclusion of living material or of pharmaceutical active principles ingels consisting of polysaccharides is therefore possible.

Macromolecular natural polysaccharides are composed of nonhydrolyzedsaccharide molecules linked to one another so as to form long chains.

The macromolecules can be composed of approximately 3 000 saccharidemolecules, and preferably of 300 to 1 200 molecules.

Various means and methods have been used to obtain alginate gels whichcan be used as an implantable material which may or may not containliving tissue extracts and/or pharmaceutical active principles.

An alginate gel composed of a suspension of hydrogel particles having adiameter of between 30 and 500 μm is, for example, known from WO98/25053.

A composite material is known, from GB 2201966, which consists of twogellable compounds, in which part of one of the two gellable compoundsis removed, thus making it possible to form a porous composite gelhaving cavities containing, for example, cells.

The influence of solutions of metal cation salts on the hardness of gelsconsisting of agar and of sodium alginate is known from the publicationHokusuishi Geppo (1967), 24, 350-6.

More recently, for applications in the food domain, the use of compoundswhose gelling process is thermo-reversible in a mixture with sodiumalginate has been revealed in JP62296853, to produce gels capable ofwithstanding high temperatures, while at the same time being relativelynon-distortable.

Macromolecular natural polysaccharides are composed of nonhydrolyzedsaccharide molecules linked to one another so as to form long chains.

The macromolecules can be composed of approximately 3 000 saccharidemolecules, and preferably of 300 to 1 200 molecules.

Among the solutions previously implemented, none have to date made itpossible to prepare implants exhibiting mechanical properties compatiblewith their use in surgical acts and easy handling.

It is, for example, known practice to use alginate gels. Alginates arederivatives of algin, which is a complex polysaccharide present in thecell walls of a brown algae belonging to the phaetophyceae group. Thealginate is extracted from the algae in the form of a sodium saltsolution.

Alginates exhibit in particular the property of forming gels by reactionwith divalent cations. These gels generally consist of approximately97.5 to 99% of water, and 0.3 to 2.5% of polymers.

The inclusion of living material or of pharmaceutical active principlesis possible; in particular, it is known practice to use alginate gels tomaintain cells in suspension. Said cells can, for example, be dispersedin a solution of alginate which will be polymerized upon contact with asolution of calcium or barium salt.

However, this method of polymerization does not make it possible to forma block of gel which can be shaped, nor does it enable molding.Independent beads of a gel which nevertheless exhibits advantageousmechanical characteristics, since it is not crumbly, it can be handled,and it can even be cut up, are obtained.

Among the other polysaccharides which can be used, there is known to beagarose, which is derived from the agar extracted from various sorts ofred algae belonging to the rhodophyceae group; it is a linearhydrocolloid galactan.

Agar and agaroses have the property of forming gels by cooling; thesegels exhibit physical properties which are variable depending on theorigin of the agars or of the agaroses. These polysaccharides cantherefore be readily shaped due to their gelling properties as afunction of temperature, for example by molding under hot conditions.They can also, by working with a solution, be molded at a temperatureabove the gelling temperature, and then, after cooling, be removed fromthe molds.

These agars or agaroses also have the particularity of exhibitinggelling temperatures of less than 40° C., compatible with cell life andbelow the temperatures of degradation of many pharmaceutical activeprinciples. However, the mechanical characteristics of these gels areinsufficient since they are crumbly and brittle and do not withstand theconstraints imposed during the performance of surgical acts.

The present invention makes it possible to produce implants exhibitingmechanical properties compatible with their use in particular duringsurgical acts and allowing chondrocyte transplantation.

The present invention also relates to a biocompatible and gellableaqueous composition of polysaccharides, characterized in that itcomprises a mixture of at least two polysaccharides, one of which ischemically gellable and the other of which is thermally gellable.

A gellable polysaccharide is thus a polysaccharide exhibiting theproperty of forming a gel by crosslinking, such as alginates, agarosesor chitosans.

The crosslinking is carried out, depending on the nature of thepolysaccharides used, by decreasing the temperature or by adding asaline solution, for example containing calcium or barium ions, or bymodifying the pH.

The term “thermally gellable” is here intended to mean the ability of acomposition to form a gel via a decrease in temperature.

The term “chemically gellable” is here intended to mean the ability of acomposition to form a gel via reaction, for example, with metal cationswhich cause crosslinking and therefore gel formation, or by modificationof the pH of the polysaccharide solution.

The chemically gellable polysaccharides according to the invention arechosen from alginates, chitosans and collagen.

Chitosans consist of N-acetylglucosamine residues linked by beta-1,4linkages, derived by deacetylation of chitin which forms long chains.Chitin is the main constituent of the exoskeleton of crustacea.

Collagen is a complex scleroprotein which is insoluble in water andsaline solutions, which is converted to gelatin in boiling water, andwhich is made up of tropocollagen macromolecules. Collagen is theessential component of the fibrils and fibers of the connective tissue.

The thermally gellable polysaccharides according to the invention areagar or agarose.

The two preferred polysaccharides according to the invention are agaroseand alginate.

The aqueous compositions of polysaccharides according to the inventioncontain between 0.5 and 10% of chemically gellable polysaccharide andbetween 0.5 and 10% of thermally gellable polysaccharide.

The present invention relates to a biocompatible and gellable aqueouscomposition of polysaccharides, one of which is chemically gellable andthe other of which is thermally gellable, this mixture making itpossible, by formation of the gel by crosslinking via a decrease intemperature, to create a moldable and solid structure, then making itpossible, by chemical crosslinking of the second polysaccharide, toimprove the mechanical properties.

The present invention relates to a biocompatible and gellable aqueouscomposition of polysaccharides, one of which is chemically gellable andthe other of which is thermally gellable, characterized in that itcomprises living biological tissue extracts or autologous, allogenic orxenogenic cells.

The cells which can be used in a composition according to the inventionbelong to the group of chondrocytes and other cells constitutingcartilage, to the group of osteoplasts or other cells constituting bone,to the group of muscle cells or fibroblasts.

These cells will preferably be chosen from cells capable of stimulatingbone or cartilaginous tissue regeneration.

The cells capable of stimulating cartilaginous tissue regeneration are,for example, chondrocytes isolated and multiplied by cell culture fromcartilage biopsy.

The biocompatible gellable compositions prepared according to theinvention can also contain demineralized spongy bone.

The biocompatible gellable compositions prepared according to theinvention can also contain agents capable of treating or preventingpathological conditions or complications.

These agents are, for example, pharmaceutical active principles such as,for example, antiviral agents or antibacterial agents.

These agents can be present in the biocompatible gellable compositionsaccording to the invention alone or as mixtures.

The compositions according to the invention are prepared using themethod comprising the following steps:

-   -   solubilization of the chemically gellable polymer in water,    -   addition of the thermally gellable polysaccharide and dissolving        by heating,    -   sterilization and cooling.

A partially crosslinked intermediate gel is produced, which isoptionally redissolved by heating before addition of the cellsuspensions or of the pharmaceutical active principles.

-   -   cooling of the mixture obtained for thermal gelling and molding,        an intermediate form is therefore obtained which does not        exhibit the mechanical properties required for the use;    -   removal from the mold and then gelling by chemical crosslinking,        by bringing into contact with a saline solution.

This method also makes it possible to obtain, if necessary, sterilizableintermediate gels which are ready to use for the incorporation of activeprinciples and/or of cell suspensions.

In a preparation variant, the chemical gelling can be carried out insitu at the time of implantation or after implantation by addition of asaline solution.

The saline solutions according to the invention are preferably solutionsof calcium or barium salts.

EXAMPLES OF USE

I. Implant Containing Chondrocytes Intended for Joint Cartilage Repair

Production of an implant containing chondrocytes

The implant is made up of cultured chondrocytes, of a composite gel and,optionally, of human demineralized spongy bone.

Preparation of the Chondrocytes

The chondrocytes originate from a cartilage biopsy performed on apatient suffering from chondral or osteochondral lesions. Afterisolation using a suitable enzyme treatment, the chondrocytes aremultiplied in cell culture until a sufficient number are obtained. Allthe steps for obtaining the chondrocytes are carried out in asepticmedium.

These cells are then recovered and suspended in culture medium beforeproduction of the implant.

Preparation of the Demineralized Spongy Bone:

The human bone tissue originates from a tissue bank. It undergoes a stepof devitalization and of viral inactivation by chemical and physicalaction. The bone tissue is adjusted to the desired form and thendemineralized in acid medium at 4° C. The sterile condition is obtainedby gamma radiation.

Preparation of the Composite Gel:

-   1. Alginate is dissolved in a Hank's solution at ambient temperature    at a concentration generally between 1 and 10%.-   2. “Low gelling” agarose is suspended in the preceding mixture at a    concentration generally between 1 and 10%.-   3. After homogenization, the mixture is immediately heated to 90° C.    in order to dissolve the agarose in suspension.-   4. The solution of alginate and agarose is sterilized by autoclaving    at 121° C. for 20 minutes.-   5. The composite gel forms after cooling to agarose gelling    temperature.

Preparation of the Implant

All of the operations are carried out under aseptic conditions

-   1. The composite gel is liquefied by heating at 90° C. and is then    kept at 37° C. for use.-   2. The cell suspension is added.-   3. After homogenization, the mixture is poured into a mold.-   4. For the implants with bony phase, the demineralized spongy bone    is then added as needed.-   5. The mold is then brought to 4° C. for 10 minutes to allow the    implant to gel.-   6. The implant is removed from the mold and is then immersed in a 25    mM CaCl₂ solution for 10 minutes in order to convert the sodium    alginate to calcium alginate.-   7. The implant is incubated at 37° C., 5% CO₂, in DMEM medium    adjusted to give 3 mM CaCl₂, until use.

Preparation Variant

When the implant does not contain demineralized bone, it is possible toconvert the sodium alginate to calcium alginate only in situ at the timeof implantation. The implant is then used as a paste which is shaped inthe lesion to be treated. Spraying a solution containing 20 mM ofcalcium chloride causes the implant to solidify in situ, adopting theshape of the lesion.

II. Femoral Obturator Intended to Facilitate Placing Pressure onSurgical Cement and to Limit its Progression Into the Diaphyseal Canalof the Femoral Shaft During Hip Replacement

The obturators conventionally used are either made of biodegradablesynthetic materials (polymer of lactic or glycolic acid), or based ongelatin of animal origin (generally porcine origin). In the first case,lysis of the polymer causes inflammatory phenomena which areundesirable. In the second case, the use of a product of animal originrepresents a potential risk of transmission of infectious diseases(prions).

Production of a Femoral Obturator

-   1. Alginate is dissolved in water for injectable preparation, at a    concentration generally between 1 and 10%.-   2. “High gelling” agarose is suspended at a concentration generally    between 1 and 10%.-   3. The mixture is immediately brought to a temperature above the    melting temperature of the agarose, in a mixer, while at the same    time homogenizing.-   4. The gel is poured into obturator molds preheated to 50° C.-   5. The molds are allowed to cool for 30 minutes at 4° C. and the gel    is then removed from the molds.-   6. The obturators are immersed in a 100 mM CaCl₂ solution for 12    hours.-   7. The obturators are rinsed for two times one hour in injectable    quality physiological saline.

1. A molded gel implant comprising a biocompatible and gellable aqueouscomposition of polysaccharides, the composition consisting of a mixtureof two polysaccharides, wherein one of the two polysaccharides ischemically gellable and the other of the two polysaccharides isthermally gellable, wherein the chemically gellable polysaccharide is acalcium alginate, wherein the thermally gellable polysaccharide isselected from the group consisting of agarose and agar, wherein thebiocompatible and gellable aqueous composition contains biologicaltissue extracts, which are autologous, allogenic or xenogenic cellsselected from the group consisting of chondrocytes, cells constitutingcartilage, osteoblasts, cells constituting bone, muscle cells andfibroblasts, and wherein the molded gel implant is obtained by thermalgelling followed by chemical gelling of the biocompatible and gellableaqueous composition.
 2. A molded gel implant comprising a biocompatibleand gellable aqueous composition of polysaccharides, the compositionconsisting of a mixture of two polysaccharides, wherein one of the twopolysaccharides is chemically gellable and the other of the twopolysaccharides is thermally gellable, wherein the chemically gellablepolysaccharide is a calcium alginate, wherein the thermally gellablepolysaccharide is selected from the group consisting of agarose andagar, wherein the biocompatible and gellable aqueous compositioncontains at least one pharmaceutically active principle chosen from thegroup consisting of antiviral agents and antibacterial agents, andwherein the molded gel implant is obtained by thermal gelling followedby chemical gelling of the biocompatible and gellable aqueouscomposition.
 3. A molded gel implant comprising a biocompatible andgellable aqueous composition of polysaccharides, the compositionconsisting of a mixture of two polysaccharides, wherein one of the twopolysaccharides is chemically gellable and the other of the twopolysaccharides is thermally gellable, wherein the chemically gellablepolysaccharide is a calcium alginate, wherein the thermally gellablepolysaccharide is selected from the group consisting of agarose andagar, wherein the biocompatible and gellable aqueous compositioncontains demineralized spongy bone, and wherein the molded gel implantis obtained by thermal gelling followed by chemical gelling of thebiocompatible and gellable aqueous composition.
 4. The molded gelimplant as claimed in claim 1, wherein the polysaccharides arerespectively present in the gel at a concentration between 0.5 and 10%.5. A method of preparing an implant, consisting essentilly of: preparinga composite gel that comprises a biocompatible and gellable aqueouscomposition of polysaccharides consisting of a mixture of twopolysaccharides, wherein one of the two polysaccharides is chemicallygellable and the other of the two polysaccharides is thermally gellable,by: solubilization of the chemically gellable polysaccharide in water,wherein the chemically gellable polysaccharide is an alginate, additionof the thermally gellable polysaccharide and dissolving by heating,wherein the thermally gellable polysaccharide is selected from the groupconsisting of agarose and agar, homogenization of the mixture, castingthe mixture into a mold, cooling of the mixture obtained for thermalgelling and molding, and removing the thermally gelled and moldedcomposition from the mold and then gelling by chemical crosslinking bybringing the thermally gelled and molded composition into contact with acalcium saline solution.
 6. A method for preparing a molded gel ofpolysaccharides, comprising: solubilization of a chemically gellablepolysaccharide in water, wherein the chemically gellable polysaccharideis an alginate, addition of a thermally gellable polysaccharide to thechemically gellable polysaccharide and dissolving the thermally gellablepolysaccharide by heating to form a mixture, wherein the thermallygellable polysaccharide is selected from the group consisting of agaroseand agar, sterilization and cooling, production of a partiallycrosslinked intermediate gel, solubilization of the intermediate gel byheating, addition of the pharmaceutically active principles selectedfrom the group consisting of antiviral agents and antibacterial agentsand/or of the biological tissue extracts, which are autologous,allogenic or xenogenic cells selected from the group consisting ofchondrocytes, cells constituting cartilage, osteoblasts, cellsconstituting bone, muscle cells and fibroblasts and/or of demineralizedspongy bone, casting the mixture into a mold, cooling of the mixtureobtained for thermal gelling and molding, removal from the mold and thengelling by chemical crosslinking, by bringing the thermally gelled andmolded composition into contact with a calcium saline solution.
 7. Amethod for preparing a femoral obturator, consisting essentially of:solubilization of a chemically gellable polysaccharide in water, whereinthe chemically gellable polysaccharide is an alginate, addition of athermally gellable polysaccharide to the chemically gellablepolysaccharide and dissolving the thermally gellable polysaccharide byheating to form a mixture, wherein the thermally gellable polysaccharideis selected from the group consisting of agarose and agar, casting themixture into a mold, cooling of the mixture obtained for thermal gellingand molding, removal from the mold and then gelling by chemicalcrosslinking, by bringing into contact with a calcium saline solution.8. A molded gel implant consisting essentially of a biocompatible andgellable aqueous composition of polysaccharides consisting of a mixtureof two polysaccharides, wherein one of the two polysaccharides ischemically gellable and the other of the two polysaccharides isthermally gellable, wherein the molded gel implant is prepared by:preparing a composite gel that comprises the biocompatible and gellableaqueous composition of polysaccharides, by: solubilization of thechemically gellable polysaccharide in water, wherein the chemicallygellable polysaccharide is an alginate, addition of the thermallygellable polysaccharide and dissolving by heating, wherein the thermallygellable polysaccharide is selected from the group consisting of agaroseand agar, homogenization of the mixture, casting the mixture into amold, cooling of the mixture obtained for thermal gelling and molding,and removing the thermally gelled and molded composition from the moldand then gelling by chemical crosslinking by bringing the thermallygelled and molded composition into contact with a calcium salinesolution to obtain the molded gel implant.
 9. A molded gel implantconsisting essentially of a biocompatible and gellable aqueouscomposition of polysaccharides, the composition consisting of a mixtureof two polysaccharides, wherein one of the two polysaccharides ischemically gellable and the other of the two polysaccharides isthermally gellable, wherein the chemically gellable polysaccharide is analginate, wherein the thermally gellable polysaccharide is selected fromthe group consisting of agarose and agar, and wherein the molded gelimplant is obtained by thermal gelling followed by chemical gelling ofthe biocompatible and gellable aqueous compositions by bringing thebiocompatible and gellable aqueous compositions into contact with acalcium saline solution.
 10. The molded gel implant as claimed in claim1, wherein the chemically gellable polysaccharide is a calcium alginateand the thermally gellable polysaccharide is an agarose.
 11. The moldedgel implant as claimed in claim 2, wherein the chemically gellablepolysaccharide is a calcium alginate and the thermally gellablepolysaccharide is agarose.
 12. The molded gel implant as claimed inclaim 3, wherein the chemically gellable polysaccharide is a calciumalginate and the thermally gellable polysaccharide is agarose.